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EP3536787A1 - Polynucléotides résistant aux nucléases et leurs utilisations - Google Patents

Polynucléotides résistant aux nucléases et leurs utilisations Download PDF

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Publication number
EP3536787A1
EP3536787A1 EP18206347.9A EP18206347A EP3536787A1 EP 3536787 A1 EP3536787 A1 EP 3536787A1 EP 18206347 A EP18206347 A EP 18206347A EP 3536787 A1 EP3536787 A1 EP 3536787A1
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EP
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Prior art keywords
mrna
polynucleotide
stabilizing oligonucleotide
polynucleotides
stabilizing
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EP18206347.9A
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German (de)
English (en)
Inventor
Michael Heartlein
Braydon Charles Guild
Frank Derosa
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Translate Bio Inc
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Translate Bio Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • exogenous nucleic acids and polynucleotides for example DNA vectors and plasmids
  • administration of such exogenous nucleic acids to a subject remains especially challenging.
  • gene therapies that rely on viruses to carry and deliver exogenous polynucleotides (e.g., DNA) to host cells and that cause the integration of such polynucleotides into the host cells' genome are capable of eliciting serious immunological and inflammatory responses.
  • the integration of such exogenous polynucleotides into the host cells' genome has the potential of misregulating the expression of the host's endogenous genes and unpredictably impacting cellular activity.
  • plasmid vector expression systems have represented an attractive alternative approach for gene therapy because of their ease of preparation, stability, and relative safety compared to viral vectors. Such plasmids are however, frequently characterized as having highly inefficient cellular uptake in vivo.
  • recombinantly-prepared proteins e.g., enzymes
  • the use of recombinant proteins and enzymes may provide a means of ameliorating the symptoms of the underlying deficiency, the utility of such therapies are often limited and are not considered curative.
  • recombinant proteins or enzymes are often prepared using non-human cell lines and may lack certain post-translational modifications (e.g., human glycosylation) relative to their endogenously produced counterparts.
  • Recombinant protein and enzyme replacement therapies are also associated with great financial expense. For example, the average cost for enzyme replacement therapy in the United States may approach $200,000-$300,000 USD per year depending on the subject's weight and prescribed dose. ( Brady, RO., Annual Review of Medicine (2006), 57: 283-296 .) Since replacement therapies are not curative, the costs associated with, for example, enzyme replacement therapies impose a significant burden on the already taxed healthcare system. Further contributing to the costs associated with such therapies, such therapies often require the administration of multiple weekly or monthly doses, with each such dose being administered by a qualified healthcare professional.
  • RNA e.g., mRNA
  • mRNA RNA
  • the administration of polynucleotides such as RNA (e.g., mRNA) that do not have to be transcribed may also represent a suitable alternative to protein or enzyme replacement therapies.
  • RNA e.g., mRNA
  • exogenous therapeutic mRNA polynucleotides encoding functional proteins or enzymes represents a promising advancement
  • the utility of such treatments may be limited by the poor stability of such polynucleotides in vivo.
  • the poor stability of exogenous polynucleotides may result in the inefficient expression (e.g., translation) of such polynucleotides, further resulting in a poor production of the protein or enzyme encoded thereby.
  • Ribonucleases represent a class of nuclease enzymes that are capable of catalyzing the degradation of RNA polynucleotides into smaller components and thereby render the polynucleotide ineffective.
  • Nuclease enzymes e.g., RNase
  • t1 ⁇ 2 the circulatory half-life of, for example, exogenous or recombinantly-prepared mRNA polynucleotides.
  • the polynucleotide is not translated, the polynucleotide is prevented from exerting an intended therapeutic benefit and its efficacy significantly reduced.
  • Novel, cost effective and therapeutically efficient approaches and therapies are still needed for the treatment of protein and enzyme deficiencies.
  • strategies and therapies which overcome the challenges and limitations associated with the administration of exogenous mRNA polynucleotides, including for example, novel methods and compositions relating to the stabilization of exogenous polynucleotides.
  • polynucleotides e.g., RNA
  • compositions that exhibit enhanced stability (e.g., increased half-life in vivo ) and nuclease resistance and which facilitate the efficient expression or production of functional proteins or enzymes.
  • the development of such stable and/or nuclease resistant compositions are necessary to overcome the limitations of conventional gene therapy and could provide viable treatments or even cures for diseases associated with the aberrant production of proteins or enzymes.
  • nuclease resistant polynucleotides and related compositions and methods generally encode functional polypeptides, proteins and/or enzymes (e.g., an mRNA polynucleotide may encode a functional urea cycle enzyme).
  • Such compositions are characterized as being more resistant to nuclease degradation relative to their unmodified or native counterparts.
  • RNA polynucleotide e.g., an RNA polynucleotide
  • the polynucleotides that are the subject of the present inventions preferably encode a functional expression product (e.g., a protein or enzyme) and may be generally characterized as comprising both a coding region and a non-coding region.
  • the methods disclosed herein generally comprise a step of contacting the non-coding region of the polynucleotide (e.g., the poly-A tail of an mRNA polynucleotide) with a complementary (e.g., a perfectly complementary) stabilizing oligonucleotide under suitable conditions, thereby causing the stabilizing oligonucleotide to hybridize to the non-coding region of the polynucleotide.
  • a complementary e.g., a perfectly complementary
  • stabilizing oligonucleotide e.g., a 15-mer poly-U oligonucleotide
  • nuclease resistance of an mRNA polynucleotide comprising a poly-A tail by contacting the poly-A tail of such polynucleotide with a complementary poly-U stabilizing oligonucleotide.
  • a complementary poly-U stabilizing oligonucleotide Upon hybridizing to the non-coding region of the polynucleotide (e.g., the poly-A tail) to form a duplexed or double-stranded region, nuclease degradation of the polynucleotide may be reduced, delayed or otherwise prevented.
  • nuclease resistance of the polynucleotides disclosed herein is due in part to the single-stranded specificity of some nuclease enzymes (e.g., ribonucleases).
  • the stabilizing oligonucleotides disclosed herein may hybridize to the non-coding region of the polynucleotide (e.g., the 5' or 3' non-coding regions of an mRNA polynucleotide) so as not to interfere with the message encoded by the coding region of such polynucleotide.
  • Stabilizing oligonucleotides may be prepared such that they are perfectly complementary to a fragment of the non-coding region (e.g., perfectly complementary to a fragment of the poly-A tail of an mRNA polynucleotide).
  • the stabilizing oligonucleotide may be complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97%, 98%, 99% or 100% complementary) to one or more non-coding regions of the polynucleotide selected from the group of regions consisting of the 3' untranslated region (UTR), the 5' untranslated region (UTR), the poly-A tail and a terminal cap.
  • the stabilizing oligonucleotide may be complementary (e.g., perfectly complementary) to a region spanning discreet structures within the non-coding region.
  • a stabilizing oligonucleotide may be prepared such that it is perfectly complementary to a region (or fragment of a region) that spans either the 3' UTR and the poly-A tail or alternatively the 5' UTR and a 5' cap structure.
  • hybridization to a region of the polynucleotide comprising fragments of both the coding and non-coding regions may provide a means to direct the hybridization of the stabilizing oligonucleotide to a specific region of the polynucleotide.
  • exogenous stabilizing oligonucleotides to a subject, for example, to treat a disease or condition associated with the aberrant expression or under-expression or production of a protein or enzyme.
  • the foregoing may be particularly suitable for the treatment of diseases or conditions characterized as having a suboptimal or sub-therapeutic endogenous production of a protein or enzyme.
  • an exogenous stabilizing oligonucleotide that is complementary e.g., perfectly complementary
  • a region of the under expressed endogenous polynucleotide e.g., one or more of the 5' and/or 3' UTR
  • such oligonucleotide may hybridize to the one or more endogenous polynucleotides (e.g., mRNA) encoding an under-expressed polypeptide, protein or enzyme such that the stability (e.g., the nuclease resistance) of the endogenous polynucleotide is modulated (e.g., enhanced or otherwise increased).
  • endogenous polynucleotides e.g., mRNA
  • the stabilized or nuclease resistant endogenous polynucleotide (e.g., mRNA) may be characterized as having an increased circulatory half-life (t1 ⁇ 2) and/or an increased translational efficiency relative to its native counterpart, generally causing the amount of the expression product (e.g., a lysosomal enzyme) encoded by such endogenous polynucleotide to be enhanced or otherwise increased.
  • the stabilizing oligonucleotide is delivered or administered in a suitable pharmaceutical carrier or composition (e.g., encapsulated in a lipid nanoparticle vehicle).
  • the present invention is directed to stable or nuclease resistant polynucleotides (e.g., mRNA) and methods of their preparation.
  • polynucleotides e.g., recombinantly-prepared mRNA
  • Such polynucleotides may be prepared by hybridizing one or more complementary (e.g., perfectly complementary) stabilizing oligonucleotides to the coding and/or non-coding regions of the polynucleotide.
  • the polynucleotides disclosed herein may encode a functional polypeptide, protein or enzyme.
  • the polynucleotide may encode a protein or enzyme selected from the group consisting of erythropoietin, human growth hormone, cystic fibrosis transmembrane conductance regulator (CFTR), alpha-galactosidase A, alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase, alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfate sulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase, hepara
  • Such methods comprise a step of administering a composition (e.g., a liposomal vehicle) comprising one or more of the nuclease resistant polynucleotides (e.g., mRNA) of the present invention to a subject affected by such disease or condition.
  • a composition e.g., a liposomal vehicle
  • the expression product e.g., a polypeptide, protein or enzyme
  • the expression product e.g., a translated protein or enzyme
  • the expression product may be excreted extracellularly by the one or more targeted host cells (e.g., hepatocytes).
  • stabilized or nuclease resistant polynucleotides e.g., mRNA
  • a complementary stabilizing oligonucleotide hybridized to the coding and/or non-coding regions of such polynucleotide.
  • the stabilizing oligonucleotide and/or the polynucleotide comprise at least one modification.
  • the modification of one or both of the polynucleotide (e.g., mRNA) and/or the stabilizing oligonucleotide to incorporate one or more modifications may be used as a means of further modulating (e.g., enhancing or increasing) the nuclease resistance of the polynucleotide.
  • modifications e.g., 2'-O-alkyl sugar modifications
  • the polynucleotide and/or the stabilizing oligonucleotide comprise at least one (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more) modified nucleobase.
  • Contemplated modifications may include, for example, sugar modifications or substitutions (e.g., one or more of a 2'-O-alkyl modification, a locked polynucleotide (LNA) or a peptide polynucleotide (PNA).)
  • sugar modifications or substitutions e.g., one or more of a 2'-O-alkyl modification, a locked polynucleotide (LNA) or a peptide polynucleotide (PNA).
  • LNA locked polynucleotide
  • PNA peptide polynucleotide
  • such modification may be selected from the group consisting of a 5-methyl cytidine, pseudouridine, 2-thio uridine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, and combinations thereof.
  • the contemplated modification may involve the inter-nucleosidic bonds that comprise the stabilizing oligonucleotide and/or the polynucleotides.
  • contemplated modifications introduced to one or both of the stabilizing oligonucleotide and/or the polynucleotide may include one or more phosphorothioate bonds.
  • all of the inter-nucleosidic bonds of one or both of the stabilizing oligonucleotide and the polynucleotide are phosphorothioate bonds.
  • the nuclease resistance of the polynucleotides disclosed herein may be characterized relative to the native or unmodified counterpart polynucleotides (e.g., relative to an un-hybridized polynucleotide that has not been contacted or treated with a stabilizing oligonucleotide).
  • the nuclease resistant polynucleotides disclosed herein may be at least about two, three, four, five, six, seven, eight, nine, ten, twelve, fifteen, twenty, twenty-five, thirty, fifty, one hundred times more stable in vivo relative to their native or un-hybridized counterparts.
  • the circulatory half-life (t1 ⁇ 2) of the polynucleotide in vivo is indicative of such polynucleotide's stability.
  • the relative amount of expression product (e.g., a polypeptide, protein or enzyme) expressed (e.g., translated) from the polynucleotide is indicative of its stability.
  • the present invention relates to methods of increasing the quantity of an expression product (e.g., a functional protein or enzyme) that is or may be expressed (e.g., translated) from a polynucleotide transcript.
  • an expression product e.g., a functional protein or enzyme
  • such methods may generally comprise a step of contacting a portion of the coding and/or non-coding regions of an mRNA polynucleotide transcript with a stabilizing oligonucleotide such that the stabilizing oligonucleotide hybridizes to the mRNA transcript.
  • the stabilizing oligonucleotide and the mRNA polynucleotide transcript are contacted at about a 0.1:1 ratio.
  • the stabilizing oligonucleotide and the mRNA polynucleotide transcript are contacted at about a 0.25:1 ratio. In yet other embodiments, the stabilizing oligonucleotide and the mRNA polynucleotide transcript are contacted at about a 0.5:1 ratio. In still other embodiments, the stabilizing oligonucleotide and mRNA polynucleotide transcript are contacted at about a 1:1 ratio. In certain embodiments, the stabilizing oligonucleotide and the mRNA polynucleotide transcript are contacted at about a 2:1, 5:1, a 10:1, a 100:1 or a 1,000:1 ratio.
  • the stabilizing oligonucleotide Upon contacting the mRNA polynucleotide transcript with a complementary stabilizing oligonucleotide, the stabilizing oligonucleotide will hybridize to the mRNA polynucleotide (e.g., at a region of complementarity). Upon hybridizing to the mRNA, the stabilizing oligonucleotide will form a duplexed region with, for example, the non-coding region of the mRNA polynucleotide and thereby render the mRNA polynucleotide more resistant to nuclease degradation.
  • the amount of the expression product (e.g., a polypeptide) translated from the mRNA polynucleotide transcript may be increased (e.g., increased by at least about 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 33%, 36%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 110%, 120%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 750%, 800%, 900%, 1,000% or more).
  • one or both of the mRNA transcript or the stabilizing oligonucleotide may comprise at least one modification (e.g., one or more chemically modified nucleobases or modified inter-nucleotide bonds).
  • Such methods may facilitate, for example, an increase in the production of an expression product produced following translation of the mRNA polynucleotides or transcripts of the present inventions.
  • Such methods comprise a step of contacting the mRNA polynucleotide transcript with a stabilizing oligonucleotide that is complementary to the coding and/or non-coding region of the mRNA transcript under suitable conditions (e.g., high stringency conditions), thereby causing the mRNA polynucleotide transcript and the stabilizing oligonucleotide to hybridize to each other.
  • the stabilizing oligonucleotide comprises at least one modified nucleobase.
  • the mRNA transcript also comprises one or more modifications (e.g., one or more chemical modifications and/or phosphorothioate inter-nucleosidic bonds).
  • the present inventions are directed to stabilized or nuclease resistant polynucleotides and compositions (e.g., mRNA polynucleotides) and related methods of their use and preparation.
  • the polynucleotides and compositions disclosed herein encode one or more functional expression products (e.g., polypeptides, proteins and/or enzymes) and are not subject to some of the limitations that are generally associated with conventional gene or enzyme replacement therapies.
  • the polynucleotide transcripts disclosed herein comprise mRNA
  • such polynucleotides need not integrate into a host cells' genome to exert their therapeutic effect.
  • the exogenous polynucleotide transcripts are translated by the host cells and accordingly are characterized by the native post-translational modifications that are present in the native expression product.
  • exogenous polynucleotides e.g., DNA or RNA
  • administration of such exogenous polynucleotides is often hampered by the limited stability of such polynucleotides, particularly following their in vivo administration.
  • many polynucleotides may be subject to nuclease (e.g., exonuclease and/or endonuclease) degradation. Nuclease degradation may negatively influence the capability of an mRNA polynucleotide transcript to reach a target cell or to be translated, the result of which is to preclude the exogenous polynucleotide from exerting an intended therapeutic effect.
  • nuclease e.g., exonuclease and/or endonuclease
  • Nucleases represent a class of enzymes that are responsible for the cleavage or hydrolysis of the phosphodiester bonds that hold the nucleotides of DNA or RNA together. Those nuclease enzymes that cleave or hydrolyze the phosphodiester bonds of DNA are called deoxyribonucleases, while the nuclease enzymes that cleave the phosphodiester bonds of RNA are called ribonucleases. As generally used herein, the term "nuclease” refers to an enzyme with the capability to degrade or otherwise digest polynucleotides or nucleic acid molecules (e.g., DNA or RNA).
  • nucleases include ribonucleases (RNase) which digests RNA, and deoxyribonuclease (DNase) which digests DNA.
  • RNase ribonucleases
  • DNase deoxyribonuclease
  • nuclease generally encompasses nuclease enzymes that are capable of degrading single-stranded polynucleotides (e.g., mRNA) and/or double stranded polynucleotides (e.g., DNA).
  • the present invention is directed to methods and strategies for stabilizing polynucleotides from nuclease degradation or for improving the resistance of one or more polynucleotides (e.g., mRNA) to nuclease degradation. It should be noted that in certain embodiments, improvements in the stability and/or nuclease resistance of the polynucleotides disclosed herein may be made with reference to a native or unmodified polynucleotide.
  • the stability and/or nuclease resistance of a polynucleotide is increased by at least about 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 150%, 160%, 170%, 175%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, or more relative to the native or unmodified polynucleotide transcript.
  • the term “stable” generally refers to a reduced susceptibility to degradation or destruction (e.g., a reduced susceptibility to nuclease cleavage in vivo ).
  • the term “stable” may be used to refer to a reduction in the rate of nuclease degradation of a polynucleotide in vivo.
  • the half-life (t1 ⁇ 2) of a polynucleotide represents an objective measurement of its stability.
  • the amount or mass of an expression product that is produced following the expression (e.g., translation) of a stable or nuclease resistant polynucleotide represents an objective measurement of its stability.
  • modifications made or otherwise introduced into a polynucleotide that serve to enhance (e.g., increase) the half-life or translational efficiency of such polynucleotide in vivo relative to its unmodified counterpart.
  • the t1 ⁇ 2 of a nuclease resistant polynucleotide is increased by at least about 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 150%, 160%, 170%, 175%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, or more relative to its native or unmodified polynucleotide counterpart.
  • the stability of hybridized mRNA may be in part due to the inherent single strand specificity of some nuclease enzymes, and in particular RNase enzymes.
  • the methods disclosed herein generally comprise a step of contacting the non-coding region of the polynucleotide (e.g., the poly-A tail of an mRNA polynucleotide) with a complementary (e.g., a perfectly complementary) stabilizing oligonucleotide under suitable conditions, thereby causing the stabilizing oligonucleotide to hybridize to the non-coding region of the polynucleotide.
  • a complementary e.g., a perfectly complementary
  • stabilizing oligonucleotide e.g., a perfectly complementary stabilizing oligonucleotide under suitable conditions
  • a polynucleotide e.g., an mRNA transcript
  • one or more stabilizing oligonucleotides e.g., a stabilizing oligonucleotide that is perfectly complementary to a region or fragment of the polynucleotide
  • the polynucleotide and stabilizing oligonucleotide would be expected to react (e.g., hybridize to one another) under suitable conditions.
  • the polynucleotide Upon hybridizing of the stabilizing oligonucleotide (e.g., a 15-mer poly(2'-O-Me-uracil) oligonucleotide) to the polynucleotide, the polynucleotide is rendered more resistant to nuclease degradation.
  • the stabilizing oligonucleotide e.g., a 15-mer poly(2'-O-Me-uracil) oligonucleotide
  • the stabilizing oligonucleotide e.g., a 15-mer poly(2'-O-Me-uracil) oligonucleotide
  • nuclease degradation of the polynucleotide may be reduced, delayed or otherwise prevented.
  • nuclease enzymes e.g., ribonucleases
  • the stabilizing oligonucleotide and/or the polynucleotide comprise a modification (e.g., a chemically-modified nucleobases and/or a phosphorothioate backbone) such modifications may serve to further stabilize the polynucleotide by sterically interfering with nuclease degradation.
  • a modification e.g., a chemically-modified nucleobases and/or a phosphorothioate backbone
  • polynucleotide and “oligonucleotide” may be generally understood by those of ordinary skill in the art to generally be synonymous with each other, such terms are used herein for convenience to distinguish the targeted sense nucleic acid transcripts (e.g., mRNA) from the shorter (e.g., about 15-50 mer) complementary or anti-sense nucleic acids that are used to modulate the stability of a targeted sense nucleic acid transcript in accordance with the teachings of the present inventions.
  • stabilizing oligonucleotide is used herein to describe a nucleic acid sequence that is generally complementary or anti-sense to a region or fragment of a polynucleotide sequence encoding a functional expression product.
  • the stabilizing oligonucleotides may generally be of any length, in certain embodiments the stabilizing oligonucleotides are less than 500 nucleotides, less than 400 nucleotides, less than 300 nucleotides, less than 250 nucleotides, less than 200 nucleotides, less than 100 nucleotides, or more preferably less than 50 nucleotides, less than 40 nucleotides, less than 30 nucleotides, less than 25 nucleotides, less than 20 nucleotides, less than 19 nucleotides, less than 18 nucleotides, less than 17 nucleotides, less than 16 nucleotides or less than 15 nucleotides in length.
  • the stabilizing oligonucleotides e.g., a 15-mer poly-U stabilizing oligonucleotide
  • the stabilizing oligonucleotide comprises one or more chemical modifications, such as one or more 2'-O-alkyl modified or substituted nucleobases or the inclusion of one or more phosphorothioate inter-nucleobase linkages.
  • Such modifications may further improve the ability of the stabilizing oligonucleotide to hybridize to a complementary polynucleotide or may improve the stability or nuclease resistance of such polynucleotide (e.g., by interfering with recognition of such polynucleotide by nuclease enzymes).
  • the present inventors have surprisingly discovered that stabilized mRNA polynucleotides that were prepared by exposure of the mRNA polynucleotides to higher concentrations of stabilizing oligonucleotides resulted in the production of lower quantities of the encoded expression product (e.g., erythropoietin protein) by cells transfected with such polynucleotides.
  • the encoded expression product e.g., erythropoietin protein
  • the longer duplexed regions may also interfere with the ability of the duplexed mRNA transcript to properly fold.
  • the proper folding of such mRNA transcript contributes to its stability (e.g., nuclease resistance)
  • it is expected that interference with the ability of such transcript to properly fold may be associated with a corresponding reduction in its stability.
  • shorter stabilizing oligonucleotides e.g., about 75, 70, 60, 65, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 nucleotides or less
  • the hybridization of a stabilizing oligonucleotide to a polynucleotide does not materially interfere with the ability of the resulting nuclease resistant polynucleotide to form secondary or tertiary structures.
  • the degree to which the nuclease resistant polynucleotides disclosed herein hybridize may be a direct function of the manner in which such nuclease resistant polynucleotides were prepared. As depicted in FIG. 1 , to the extent that an mRNA polynucleotide is contacted with a high concentration of a complementary stabilizing oligonucleotide, the stabilizing oligonucleotide may hybridize to the mRNA polynucleotide at multiple regions.
  • the extent to which a polynucleotide hybridizes with a complementary stabilizing oligonucleotide may be manipulated or otherwise controlled by modifying the relative concentrations of stabilizing oligonucleotide to which the polynucleotide is exposed.
  • the stabilizing oligonucleotide and the mRNA polynucleotide transcript are contacted at about a 0.1:1 ratio. In other embodiments, the stabilizing oligonucleotide and the mRNA polynucleotide transcript are contacted at about a 0.25:1 ratio.
  • the stabilizing oligonucleotide and the mRNA polynucleotide transcript are contacted at about a 0.5:1 ratio. In still other embodiments, the stabilizing oligonucleotide and mRNA polynucleotide transcript are contacted at about a 1:1 ratio. In certain embodiments, the stabilizing oligonucleotide and the mRNA polynucleotide transcript are contacted at about a 5:1, a 10:1, a 100:1 or a 1,000:1 ratio.
  • polynucleotide is generally used to refer to a nucleic acid (e.g., DNA or RNA) to be stabilized or rendered more nuclease resistant in accordance with the teachings of the present invention.
  • the polynucleotides disclosed herein represent the nucleic acid target to which the stabilizing oligonucleotides may hybridize.
  • the polynucleotides (e.g., an mRNA polynucleotide) disclosed herein may also comprise one or more modifications.
  • the mRNA polynucleotide transcripts disclosed herein comprise one or more chemical modifications, which in certain instances may further improve the stability or nuclease resistance of such polynucleotide transcript (e.g., by sterically hindering or otherwise interfering with nuclease degradation).
  • the polynucleotides may comprise both coding and non-coding regions and in certain embodiments described herein, the stabilizing oligonucleotides hybridize to the non-coding region of the polynucleotide.
  • non-coding region generally refers to that portion or region of the polynucleotide or a gene that is not a coding region and that is not expressed, transcribed, translated or otherwise processed into an expression product such as an amino acid, polypeptide, protein or enzyme.
  • the non-coding region may comprise intron sequences or other sequences located 5' or 3' (e.g., upstream or downstream) of the coding region (e.g., promoters, enhancers, silencers).
  • the non-coding region may comprise sequences located 5' or 3' (e.g., upstream or downstream) of the coding region (e.g., 3' untranslated region (UTR), a 5' untranslated region (UTR), a poly-A tail and a terminal cap).
  • the targeted non-coding region may comprise two distinct, but overlapping regions.
  • a stabilizing oligonucleotide may be prepared such that it is perfectly complementary to a region of a polynucleotide comprising or spanning a fragment of the 3' untranslated region (UTR) and a fragment of the poly-A tail.
  • mRNA Polynucleotide Fragment Stabilizing Oligonucleotide:
  • a stabilizing oligonucleotide may be prepared such that it is complementary to a region of a polynucleotide comprising or spanning a fragment of a 5' cap structure and a fragment of the 5' UTR.
  • the stabilizing oligonucleotide may be complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97%, 98%, 99% or 100% complementary) to one or more non-coding regions of the polynucleotide selected from the group of regions consisting of the 5' UTR, a 5' terminal cap, the 3'UTR and the poly-A tail.
  • the hybridization of a complementary stabilizing oligonucleotide to the non-coding region of an mRNA polynucleotide is preferred, in part due to concerns relating to the ability of the resultant duplexed region (i.e., the hybridized polynucleotide and stabilizing oligonucleotide) to interfere with the translation of the coding region.
  • coding region generally refers to that portion or region of the polynucleotide or a gene that when expressed, transcribed, translated or otherwise processed results in the production of an expression product, such as an amino acid, polypeptide, protein or enzyme. It should be understood that while certain embodiments disclosed herein contemplate the hybridization of complementary stabilizing oligonucleotides to the non-coding region of a polynucleotide transcript, the present invention need not be limited to such embodiments. Rather, the present invention also contemplates the hybridization of the complementary stabilizing oligonucleotides to regions of the polynucleotide transcript (e.g., mRNA) comprising or spanning both the coding and non-coding regions.
  • regions of the polynucleotide transcript e.g., mRNA
  • a stabilizing oligonucleotide may be prepared such that it targets and/or is complementary (e.g., perfectly complementary) to a fragment of the coding region of an mRNA polynucleotide transcript and a fragment of the non-coding 3' UTR located downstream of the coding region.
  • the foregoing therefore provides a means of specifically targeting a particular region of the polynucleotide, such as the region located immediately downstream of the coding region. Additionally, the foregoing also provides means of controlling or otherwise affecting the degree to which a stabilizing oligonucleotide hybridizes to a complementary region of the polynucleotide.
  • the stabilizing oligonucleotide targets the coding region (or a fragment thereof) preferably the hybridization of the stabilizing oligonucleotide to such coding region (or fragment thereof) does not interfere with the expression (e.g., transcription or translation) of such polynucleotide.
  • the stabilizing oligonucleotide targets the coding region (or a fragment thereof) preferably the hybridization of the stabilizing oligonucleotide to such coding region (or fragment thereof) does not substantially interfere with the expression (e.g., transcription or translation) of such polynucleotide.
  • the term "expression” is used in its broadest sense to refer to either the transcription of a specific polynucleotide (e.g., a gene or nucleic acid) into an RNA transcript, or the translation of at least one mRNA polynucleotide into a polypeptide, protein or enzyme.
  • a specific polynucleotide e.g., a gene or nucleic acid
  • mRNA polynucleotide e.g., a gene or nucleic acid
  • mRNA polynucleotide e.g., a gene or nucleic acid
  • expression refers to the translation of such mRNA polynucleotides to produce a polypeptide, protein or enzyme encoded thereby.
  • the phrase "expression product" is used herein in its broadest sense to generally refer to an RNA transcription product that is transcribed from a DNA polynucleotide, or alternatively to a polypeptide, protein or enzyme that is the natural translation product of an mRNA polynucleotide.
  • the expression product of the polynucleotide is a functional enzyme (e.g., a urea cycle enzyme).
  • the expression product of the polynucleotide is a functional protein (e.g., hormone) or enzyme.
  • the encoded expression product i.e., RNA
  • the polynucleotide is mRNA
  • following expression (i.e., translation) of such mRNA the encoded expression product (e.g., a polypeptide, protein or enzyme) may be produced and/or excreted.
  • the present inventions are directed to methods of modulating (e.g., increasing, improving or otherwise enhancing) the translational efficiency of one or more mRNA polynucleotides in a target cell.
  • translational efficiency refers to the rate at which an mRNA polynucleotide is translated and the corresponding expression product produced.
  • the stable or nuclease resistant mRNA polynucleotides disclosed herein may be characterized by their increased translational efficiency, resulting in a corresponding increase in the production of the expression product encoded by such mRNA polynucleotide.
  • Such methods generally comprise an initial step of contacting an mRNA polynucleotide with a complementary (e.g., perfectly or partially complementary) stabilizing oligonucleotide under suitable conditions (e.g., high stringency conditions), thereby causing the mRNA polynucleotide and one or more stabilizing oligonucleotides to hybridize to each other.
  • a complementary e.g., perfectly or partially complementary
  • suitable conditions e.g., high stringency conditions
  • one or both of the stabilizing oligonucleotide and/or the mRNA polynucleotide may comprise at least one modified nucleobase (e.g., a 2'-O-alkyl sugar substitution).
  • one or both of the stabilizing oligonucleotide and the mRNA polynucleotide also comprise one or more modifications (e.g., one or more nucleobases linked by phosphorothioate bonds).
  • the nuclease resistant polynucleotides disclosed herein may be recombinantly-prepared (e.g., a recombinantly-prepared codon-optimized mRNA polynucleotide).
  • such polynucleotides e.g., a recombinantly-prepared mRNA polynucleotide
  • a complementary stabilizing oligonucleotide prior to being administered to a subject in a suitable carrier or vehicle (e.g., a lipid nanoparticle).
  • an exogenous stabilizing oligonucleotide to a subject (e.g., for the treatment of a disease or condition associated with the suboptimal or sub-therapeutic production of an expression product, such as a protein or enzyme).
  • the present inventions provide a means of modulating (e.g., increasing or otherwise enhancing) the expression, production and/or secretion of an endogenous expression product.
  • the present inventions contemplate the administration of a stabilizing oligonucleotide to a subject, wherein the stabilizing oligonucleotide is complementary (e.g., perfectly- or partially-complementary) to an endogenous polynucleotide (e.g., mRNA).
  • an exogenously-prepared stabilizing oligonucleotide that is complementary (e.g., perfectly complementary) to a region of an endogenous polynucleotide (e.g., the non-coding region of an endogenous mRNA polynucleotide) is administered to a subject.
  • the exogenous stabilizing oligonucleotide hybridizes to the one or more endogenous polynucleotides (e.g., mRNA) encoding an under-expressed expression product such that the stability or the nuclease resistance of the endogenous polynucleotide is modulated (e.g., enhanced or otherwise increased) and/or its translational efficiency increased.
  • the resulting stabilized or nuclease resistant endogenous polynucleotide e.g., mRNA
  • the amount of the expression product (e.g., a lysosomal enzyme) encoded by such endogenous polynucleotides may be enhanced or otherwise increased and an underlying condition (e.g., a protein or enzyme deficiency) or its symptoms thereby treated or mitigated.
  • an underlying condition e.g., a protein or enzyme deficiency
  • the foregoing therefore provides a means of increasing the expression of sub-optimally expressed endogenous mRNA polynucleotides by rendering such polynucleotides more nuclease resistant relative to their native (and under-expressed) counterparts.
  • the foregoing embodiments may generally relate to traditional anti-sense or RNAi mechanisms of targeting endogenous nucleic acids (e.g., mRNA), the observed effect of such targeting is an increase, rather than a decrease, in the production of the expression product encoded by the targeted polynucleotide.
  • the stabilizing oligonucleotide is delivered or administered to a subject in a suitable pharmaceutical carrier, vehicle or composition (e.g., encapsulated in a lipid nanoparticle vehicle).
  • a subject e.g., mRNA which has been stabilized against in vivo nuclease digestion or degradation.
  • the therapeutic activity of the nuclease resistant polynucleotide is prolonged or otherwise evident over an extended period of time (e.g., at least about twelve hours, twenty-four hours, thirty-six hours, seventy-two hours, four days, five days, 1 week, ten days, two weeks, three weeks, four weeks, six weeks, eight weeks, ten weeks, twelve weeks or longer).
  • the therapeutic activity of the nuclease resistant polynucleotides may be prolonged such that the compositions of the present invention are administered to a subject on a semi-weekly or bi-weekly basis, or more preferably on a monthly, bimonthly, quarterly or even on an annual basis.
  • compositions of the present invention and in particular of the nuclease resistant mRNA polynucleotides comprised therein, is directly related to the translational efficiency of such polynucleotide and the quantity of the expression product (e.g., a functional protein or enzyme) that can be translated from such mRNA.
  • the expression product e.g., a functional protein or enzyme
  • the translational efficiency and the in vivo activity of the nuclease resistant polynucleotides and compositions of the present invention may be further extended or prolonged by the introduction of one or more modifications to such polynucleotides to improve or enhance their half-life (t1 ⁇ 2).
  • the Kozac consensus sequence plays a role in the initiation of protein translation, and the inclusion of such a Kozac consensus sequence in the mRNA polynucleotides of the present invention may further extend or prolong the activity or translational efficiency of such mRNA polynucleotides.
  • the quantity of functional protein or enzyme translated by the target cell is a function of the quantity of polynucleotide (e.g., mRNA) delivered to the target cells and the stability of such polynucleotide.
  • polynucleotide e.g., mRNA
  • the stability and/or half-life of the nuclease resistant polynucleotides of the present invention may be improved or enhanced, the therapeutic activity of the translated protein or enzyme and/or the dosing frequency of the composition may be further extended.
  • one or both of the polynucleotides and/or the stabilizing oligonucleotides disclosed herein comprise at least one modification.
  • the terms "modification” and “modified” as they relate to the polynucleotides and/or stabilizing oligonucleotides provided herein refer to at least one alteration or chemical modification introduced into such polynucleotides and/or stabilizing oligonucleotides and which preferably renders them more stable (e.g., resistant to nuclease digestion) than the wild-type or naturally occurring version of the polynucleotide.
  • the introduction of chemical modifications into one or more of the polynucleotide and the stabilizing oligonucleotide may interfere with, sterically hinder or otherwise delay their recognition and/or degradation by one or more nuclease enzymes (e.g., RNase).
  • Increased stability can include, for example, less sensitivity to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue, thereby increasing or enhancing the circulatory half-life or residence time of such polynucleotides in the target cell, tissue, subject and/or cytoplasm.
  • the stabilized or nuclease resistant polynucleotides provided herein may demonstrate longer half-lives relative to their naturally occurring or un-hybridized counterparts (e.g. the wild-type version of the polynucleotide).
  • modification and “modified”, as such terms relate to mRNA polynucleotides and/or stabilizing oligonucleotides of the present invention, are alterations which improve or enhance the translational efficiency of such mRNA polynucleotides, including for example, the inclusion of sequences which affect the initiation of protein translation (e.g., the Kozac consensus sequence). (See, Kozak, M., Nucleic Acids Res. (1987); 15 (20): 8125-48 ).
  • Exemplary modifications to a polynucleotide may also include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base.
  • the phrase "chemical modifications" as used herein includes modifications which introduce chemistries that differ from those observed in naturally occurring polynucleotides, for example, covalent modifications such as the introduction of modified bases (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such polynucleotides).
  • exemplary chemical modifications that may be introduced into one or both of the polynucleotide and the stabilizing oligonucleotide include pseudouridine, 2-thiouracil, 5-methyl cytidine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine.
  • suitable modifications may include alterations in one or more nucleotides of a codon such that the codon encodes the same amino acid but is more stable relative to the wild-type codon of the polynucleotide found in nature.
  • C cytidines
  • U uridines
  • the number of C and/or U residues is reduced by substitution of one codon encoding a particular amino acid for another codon encoding the same or a related amino acid.
  • Contemplated modifications to the mRNA polynucleotides of the present invention also include the incorporation of pseudouridines.
  • the incorporation of pseudouridines into the mRNA polynucleotides of the present invention may enhance their stability and translational capacity, as well as diminish their immunogenicity in vivo. (See, e.g., Karikó, K., et al., Molecular Therapy 16 (11): 1833-1840 (2008 )).
  • Substitutions and modifications to the polynucleotides of the present invention may be performed by methods readily known to one or ordinary skill in the art.
  • the constraints on reducing the number of C and U residues in a sequence will likely be greater within the coding region of an mRNA polynucleotide, compared to its untranslated region, (i.e., it will likely not be possible to eliminate all of the C and U residues present in the coding region while still retaining the ability of the message to encode the desired amino acid sequence).
  • the degeneracy of the genetic code presents an opportunity to allow the number of C and/or U residues that are present in the sequence to be reduced, while maintaining the same coding capacity (i.e., depending on which amino acid is encoded by a codon, several different possibilities for modification of RNA sequences may be possible).
  • the codons for Gly can be altered to GGA or GGG instead of GGU or GGC.
  • modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the polynucleotides and/or stabilizing oligonucleotides of the present invention.
  • modifications include the addition of bases to a polynucleotide sequence (e.g., the inclusion of a poly-A tail or the lengthening of the poly-A tail), the alteration of the 3' UTR or the 5' UTR, and the inclusion of elements which change the structure of a polynucleotide and/or stabilizing oligonucleotide (e.g., elements which modulate the ability of such polynucleotides or their expression products to form secondary structures).
  • the poly-A tail and the region immediately upstream represent suitable regions of a polynucleotide that the stabilizing oligonucleotides (e.g., a 15-mer poly-U stabilizing oligonucleotide) disclosed herein may target and/or hybridize to.
  • the poly-A tail is thought to naturally stabilize natural mRNA polynucleotides and synthetic sense RNA. Therefore, in certain embodiments a long poly-A tail can be added to an mRNA polynucleotide and thus render the mRNA more stable.
  • the poly-A tail or a particular region thereof may be contacted under suitable condition (e.g., high stringency conditions) with a complementary stabilizing oligonucleotide (e.g., a poly-U stabilizing oligonucleotide) and thereby render the polynucleotide more nuclease resistant.
  • suitable condition e.g., high stringency conditions
  • a complementary stabilizing oligonucleotide e.g., a poly-U stabilizing oligonucleotide
  • Poly-A tails can be added using a variety of art-recognized techniques. For example, long poly-A tails can be added to synthetic or in vitro transcribed RNA using poly-A polymerase. ( Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256 ).
  • poly-A tails can be added by transcription directly from PCR products or may be ligated to the 3' end of an mRNA polynucleotide with RNA ligase.
  • RNA ligase See, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 editi on)).
  • the length of the poly-A tail is at least about 20, 40, 50, 75, 90, 100, 150, 200, 250, 300, 350, 400, 450 or at least 500 nucleotides.
  • the length of the poly-A tail is adjusted to control the stability of an mRNA polynucleotide of the invention.
  • the length of the poly-A tail can influence the half-life of an mRNA polynucleotide
  • the length of the poly-A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control its translational efficiency in a target cell.
  • the stabilized or nuclease resistant polynucleotides are sufficiently resistant to in vivo degradation (e.g., by nucleases), such that they may be delivered to the target cell without a carrier.
  • a polynucleotide can be modified by the incorporation 3' and/or 5' untranslated (UTR) sequences which are not naturally found in the wild-type polynucleotide.
  • UTR untranslated
  • 3' and/or 5' flanking sequences which naturally flank an mRNA and encode a second, unrelated protein can be incorporated into the nucleotide sequence of an mRNA polynucleotide in order to further enhance its translational efficiency.
  • 3' or 5' sequences from mRNA polynucleotides which are stable can be incorporated into the 3' and/or 5' region of a sense mRNA polynucleotide to increase its stability.
  • the regions of the polynucleotide including such modifications may also represent a suitable target to which the stabilizing oligonucleotides disclosed herein may hybridize to in an effort to further stabilize such modified polynucleotide.
  • the present inventions also contemplate modifications to the 5' end of the polynucleotides (e.g., mRNA) to include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) to improve the nuclease resistance and/or improve the half-life of the polynucleotide.
  • a CMV immediate-early 1 (IE1) gene e.g., SEQ ID NO: 1 or SEQ ID NO: 2
  • hGH human growth hormone
  • SEQ ID NO: 3 a sequence encoding human growth hormone (hGH)
  • SEQ ID NO: 3 a fragment thereof (e.g., SEQ ID NO: 3)
  • preferred modifications improve the stability, translational efficiency, nuclease resistance and/or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to its unmodified counterpart, and include, for example modifications made to improve such polynucleotide's resistance to in vivo nuclease digestion.
  • compositions, stabilized polynucleotides and stabilizing oligonucleotides disclosed herein may be facilitated by formulating such compositions in a suitable carrier (e.g., a lipid nanoparticle).
  • a suitable carrier e.g., a lipid nanoparticle.
  • carrier includes any of the standard pharmaceutical carriers, vehicles, diluents, excipients and the like which are generally intended for use in connection with the administration of biologically active agents, including polynucleotides.
  • the compositions and in particular the carriers described herein are capable of delivering polynucleotides and/or stabilizing oligonucleotides of varying sizes to their target cells or tissues.
  • the carriers of the present invention are capable of delivering large polynucleotide sequences (e.g., polynucleotides of at least 1kb, 1.5kb, 2 kb, 2.5kb, 5kb, 10kb, 12kb, 15kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, or more).
  • the polynucleotides can be formulated with one or more acceptable reagents to facilitate the delivery of such polynucleotides to target cells.
  • Appropriate reagents are generally selected with regards to a number of factors, which include, among other things, the biological or chemical properties of the polynucleotides (e.g., charge), the intended route of administration, the anticipated biological environment to which such polynucleotides will be exposed and the specific properties of the intended target cells.
  • carriers such as liposomes or synthetically-prepared exosomes, encapsulate the polynucleotides.
  • the carrier demonstrates preferential and/or substantial binding to a target cell relative to non-target cells.
  • the carrier delivers its contents to the target cell such that the polynucleotides are delivered to the appropriate subcellular compartment, such as the cytoplasm.
  • the carriers disclosed herein comprise a liposomal vesicle, or other means to facilitate the transfer of a polynucleotide to target cells and tissues.
  • Suitable carriers include, but are not limited to, liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides and other vectorial tags.
  • bionanocapsules and other viral capsid proteins assemblies are also contemplated.
  • the carrier is formulated as a lipid nanoparticle.
  • lipid nanoparticle refers to a carrier comprising one or more lipids (e.g., cationic and/or non-cationic lipids).
  • the lipid nanoparticles are formulated to deliver one or more polynucleotides (e.g., mRNA) to one or more target cells or tissues.
  • polynucleotides e.g., mRNA
  • lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Also contemplated is the use of polymers as carriers, whether alone or in combination with other carriers.
  • phosphatidyl compounds e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • polymers as carriers, whether alone or in combination with other carriers.
  • Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins and polyethylenimine.
  • the carrier is selected based upon its ability to facilitate the transfection of a target cell with one or more polynucleotides.
  • the carrier may be selected and/or prepared to optimize delivery of the polynucleotide to the target cell, tissue or organ.
  • the properties of the carrier e.g., size, charge and/or pH
  • the target tissue is the central nervous system (e.g., to facilitate delivery of mRNA polynucleotides to targeted brain or spinal tissue) selection and preparation of the carrier must consider penetration of, and retention within the blood brain barrier and/or the use of alternate means of directly delivering such carrier to such target tissue.
  • the compositions of the present invention may be combined with agents that facilitate the transfer of exogenous polynucleotides from the local tissues or organs into which such compositions were administered to one or more peripheral target organs or tissues.
  • Liposomes e.g., liposomal lipid nanoparticles
  • Liposomes are generally useful in a variety of applications in research, industry, and medicine, particularly for their use as carriers of diagnostic or therapeutic compounds in vivo ( Lasic, Trends Biotechnol., 16: 307-321, 1998 ; Drummond et al., Pharmacol. Rev., 51: 691-743, 1999 ) and are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers.
  • Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains ( Lasic, Trends Biotechnol., 16: 307-321, 1998 ). Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).
  • a liposomal carrier typically serves to transport the polynucleotide and/or stabilizing oligonucleotide to a target cell.
  • the liposomal carriers are prepared to contain the desired polynucleotides.
  • the process of incorporating a desired compound (e.g., a stabilized or nuclease resistant polynucleotide and/or a stabilizing oligonucleotide) into a liposome is often referred to as "loading" ( Lasic, et al., FEBS Lett., 312: 255-258, 1992 ).
  • the liposome-incorporated polynucleotides may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane.
  • the incorporation of a polynucleotide into liposomes is also referred to herein as "encapsulation" wherein the polynucleotide is entirely contained within the interior space of the liposome.
  • a polynucleotide into a carrier such as a liposome
  • a carrier such as a liposome
  • the selected carrier is capable of further enhancing the stability of the nuclease resistant polynucleotides (e.g., a nuclease resistant mRNA polynucleotide encoding a functional protein) contained therein.
  • a liposomal carrier may allow the encapsulated polynucleotide to reach the target cell and/or may preferentially allow the encapsulated polynucleotide to reach the target cell, or alternatively limit the delivery of such polynucleotides to other sites or cells where the presence of the administered polynucleotide may be useless or undesirable.
  • incorporating the polynucleotides into a carrier such as for example, a cationic liposome, also facilitates the delivery of such polynucleotides into a target cell.
  • liposomal carriers are prepared to encapsulate one or more desired polynucleotides (e.g., a nuclease resistant mRNA polynucleotide encoding a urea cycle enzyme) such that the compositions demonstrate a high transfection efficiency, enhanced stability and improved translational efficiency.
  • desired polynucleotides e.g., a nuclease resistant mRNA polynucleotide encoding a urea cycle enzyme
  • liposomes can facilitate the introduction of polynucleotides into target cells
  • polycations e.g., poly L-lysine and protamine
  • as a copolymer can further facilitate, and in some instances markedly enhance the transfection efficiency of several types of cationic liposomes by 2-28 fold in a number of cell lines both in vitro and in vivo.
  • the present invention contemplates the use of cationic lipids and liposomes to encapsulate and/or enhance the delivery of the nuclease resistant polynucleotides and/or stabilizing oligonucleotides disclosed herein into their target cells and tissues.
  • cationic lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.
  • the contemplated liposomal carriers and lipid nanoparticles may be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and PEG-modified lipids.
  • the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA" is used.
  • DOTMA can be formulated alone or can be combined with a neutral lipid, such as, e.g., dioleoylphosphatidylethanolamine or "DOPE" or other cationic or non-cationic lipids into a liposomal carrier or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of polynucleotides into target cells.
  • a neutral lipid such as, e.g., dioleoylphosphatidylethanolamine or "DOPE” or other cationic or non-cationic lipids into a liposomal carrier or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of polynucleotides into target cells.
  • a neutral lipid such as, e.g., dioleoylphosphatidylethanolamine or "DOPE" or other cationic or non-cationic lipids into a liposomal carrier or a lipid nanoparticle,
  • Another particularly suitable cationic lipid for use in connection with the invention is 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine or "DLin-KC2-DMA" (See, WO 2010/042877 ; Semple et al., nature Biotech. 28:172-176 (2010 ).
  • Suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or "DOGS,” 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium or "DOSPA" ( Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989 ); U.S. Pat. No. 5,171,678 ; U.S. Pat. No.
  • Contemplated cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N,N-dimethylammonium bromide or "DDAB", N-(1,2-d
  • cholesterol-based cationic lipids are also contemplated by the present invention.
  • Such cholesterol-based cationic lipids can be used, either alone or in combination with other cationic or non-cationic lipids.
  • Suitable cholesterol-based cationic lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine ( Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991 ); Wolf et al. BioTechniques 23, 139 (1997 ); U.S. Pat. No. 5,744,335 ).
  • LIPOFECTIN DOTMA:DOPE
  • DOSPA:DOPE LIPOFECTAMINE
  • LIPOFECTAMINE2000. Invitrogen
  • FUGENE FUGENE
  • TRANSFECTAM DOGS
  • EFFECTENE EFFECTENE
  • cationic lipids such as the dialkylamino-based, imidazole-based, and guanidinium-based lipids.
  • certain embodiments are directed to a composition comprising one or more imidazole-based cationic lipids, for example, the imidazole cholesterol ester or "ICE" lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate.
  • imidazole-based cationic lipids for example, the imidazole cholesterol ester or "ICE" lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13,
  • a carrier for delivery of RNA (e.g., mRNA) or protein (e.g., an enzyme), for example a therapeutic amount of RNA or protein, may comprise one or more imidazole-based cationic lipids, for example, the imidazole cholesterol ester or "ICE" lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate.
  • ICE imidazole cholesterol ester or "ICE" lipid
  • the imidazole-based cationic lipids are also characterized by their reduced toxicity relative to other cationic lipids.
  • the imidazole-based cationic lipids e.g., ICE
  • the cationic lipid may comprise a molar ratio of about 1% to about 90%, about 2% to about 70%, about 5% to about 50%, about 10% to about 40% of the total lipid present in the carrier, or preferably about 20% to about 70% of the total lipid present in the carrier.
  • certain embodiments are directed to lipid nanoparticles comprising the HGT4003 cationic lipid 2-((2,3-Bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethanamine, as further described in U.S. Provisional Application No: 61/494,882 filed June 8, 2011 , the entire teachings of which are incorporated herein by reference in their entirety.
  • compositions and methods described herein are directed to lipid nanoparticles comprising one or more ionizable cationic lipids, such as, for example, one or more of the cationic lipids or compounds (e.g., HGT5001, HGT5002 and HGT5003), as further described in U.S. Provisional Application No: 61/617,468 , incorporated herein by reference in their entirety.
  • ionizable cationic lipids such as, for example, one or more of the cationic lipids or compounds (e.g., HGT5001, HGT5002 and HGT5003), as further described in U.S. Provisional Application No: 61/617,468 , incorporated herein by reference in their entirety.
  • compositions and methods described herein are directed to lipid nanoparticles comprising one or more cleavable lipids, such as, for example, one or more cationic lipids or compounds that comprise a cleavable disulfide (S-S) functional group (e.g., HGT4001, HGT4002, HGT4003, HGT4004 and HGT4005), as further described in U.S. Provisional Application No: 61/494,882 , incorporated herein by reference in their entirety.
  • S-S cleavable disulfide
  • PEG polyethylene glycol
  • PEG-CER derivatized cerarmides
  • C8 PEG-2000 ceramide C8 PEG-2000 ceramide
  • Contemplated PEG-modified lipids include, but is not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C 20 length.
  • the addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-polynucleotide composition to the target tissues, ( Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237 ), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613 ).
  • Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C 14 or C 18 ).
  • the PEG-modified phospholipid and derivatized lipids of the present invention may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposomal carrier.
  • the PEG-modified lipid employed in the compositions and methods of the invention is 1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (2000 MW PEG) (DMG-PEG2000).
  • non-cationic lipids to facilitate delivery of the nuclease resistant polynucleotides or stabilizing oligonucleotides to one or more target cells, organs or tissues.
  • non-cationic lipid refers to any neutral, zwitterionic or anionic lipid.
  • anionic lipid refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH.
  • Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE),
  • non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids.
  • the non-cationic lipid may comprise a molar ratio of 5% to about 90%, or preferably about 10 % to about 70% of the total lipid present in the carrier.
  • the carrier e.g., a lipid nanoparticle
  • a carrier may be prepared using DSPC/CHOL/DODAP/C8-PEG-5000 ceramide in a molar ratio of about 1 to 50 : 5 to 65 : 5 to 90 : 1 to 25, respectively.
  • a carrier may be comprised of additional lipid combinations in various ratios, including for example, DSPC/CHOL/DODAP/mPEG-5000 (e.g., combined at a molar ratio of about 33:40:25:2), DSPC/CHOL/DODAP/C8 PEG-2000-Cer (e.g., combined at a molar ratio of about 31:40:25:4), POPC/DODAP/C8-PEG-2000-Cer (e.g., combined at a molar ratio of about 75-87:3-14:10) or DSPC/CHOL/DOTAP/C8 PEG-2000-Cer (e.g., combined at a molar ratio of about 31:40:25:4).
  • DSPC/CHOL/DODAP/mPEG-5000 e.g., combined at a molar ratio of about 33:40:25:2
  • DSPC/CHOL/DODAP/C8 PEG-2000-Cer e
  • cationic lipids non-cationic lipids and/or PEG-modified lipids which comprise the liposomal carrier or lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells or tissues and the characteristics of the polynucleotides to be delivered by the liposomal carrier. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s).
  • the liposomal carriers for use in the present invention can be prepared by various techniques which are presently known in the art.
  • Multi-lamellar vesicles may be prepared by conventional techniques, for example, by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then added to the vessel with a vortexing motion which results in the formation of MLVs.
  • Unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multi-lamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.
  • the compositions comprise a carrier wherein a nuclease resistant polynucleotide (e.g., mRNA encoding OTC) is associated on both the surface of the carrier (e.g., a liposome) and encapsulated within the same carrier.
  • a nuclease resistant polynucleotide e.g., mRNA encoding OTC
  • cationic liposomal carriers may associate with the polynucleotides (e.g., mRNA) through electrostatic interactions with such therapeutic mRNA.
  • compositions or polynucleotides of the present invention may comprise or be loaded with a diagnostic radionuclide, fluorescent material or other material that is detectable in both in vitro and in vivo applications.
  • suitable diagnostic materials for use in the present invention may include Rhodamine-dioleoylphosphatidylethanolamine (Rh-PE), Green Fluorescent Protein mRNA (GFP mRNA), Renilla Luciferase mRNA and Firefly Luciferase mRNA.
  • water soluble carrier agents may be encapsulated in the aqueous interior by including them in the hydrating solution, and lipophilic molecules may be incorporated into the lipid bilayer by inclusion in the lipid formulation.
  • lipophilic molecules e.g., cationic or anionic lipophilic polynucleotides
  • loading of the polynucleotide into preformed liposomes may be accomplished, for example, by the methods described in U.S. Pat. No. 4,946,683 , the disclosure of which is incorporated herein by reference.
  • the liposomes may be processed to remove unencapsulated mRNA through processes such as gel chromatography, diafiltration or ultrafiltration. For example, if it is desirous to remove externally bound polynucleotide from the surface of the liposomal carrier formulation, such liposomes may be subject to a Diethylaminoethyl SEPHACEL column.
  • one or more secondary therapeutic or diagnostic agents may be included in the carrier.
  • additional therapeutic agents may be associated with the surface of the liposome, can be incorporated into the lipid bilayer of a liposome by inclusion in the lipid formulation or loading into preformed liposomes.
  • the extrusion method is a preferred method of liposome sizing. ( Hope, M J et al. Reduction of Liposome Size and Preparation of Unilamellar Vesicles by Extrusion Techniques. In: Liposome Technology (G. Gregoriadis, Ed.) Vol. 1. p 123 (1993 )).
  • the method comprises a step of extruding liposomes through a small-pore polycarbonate membrane or an asymmetric ceramic membrane to reduce liposome sizes to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved.
  • the liposomes may be extruded through successively smaller pore membranes to achieve gradual reduction in liposome size.
  • the size of the liposomal vesicles may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-450 (1981 ), incorporated herein by reference. Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.
  • QELS quasi-electric light scattering
  • Selection of the appropriate size of a carrier must take into consideration the site of the target cell or tissue and to some extent the application for which the liposome is being made.
  • the ability of the carrier to distribute into the tissues of the lung may be influenced by the size of the carrier particles that comprise such composition. Accordingly, in certain embodiments, it may be desirable to enhance the distribution of such compositions to certain cells or tissues of the lung by appropriately sizing such compositions such that upon administration (e.g., by inhalation), such compositions distribute to one or more targeted cells and tissues.
  • the compositions provided herein are generally administered via the pulmonary route of administration. Accordingly, in certain embodiments the carriers and/or compositions disclosed herein are prepared for pulmonary administration.
  • a pulmonary surfactant may be added as an excipient component of a carrier formulation (e.g., a lipid nanoparticle comprising one or more cationic lipids, neutral lipids and pulmonary surfactants).
  • the compositions disclosed herein may comprise one or more pulmonary surfactants that may be formulated independently of the carrier.
  • pulmonary surfactants e.g., lamellar bodies
  • lamellar bodies may also serve to loosen, break-up or otherwise facilitate the elimination of mucous from the lungs of the subject, thereby improving the distribution of the compositions into the tissues of the lung.
  • such lamellar bodies may also function as a carrier to facilitate the delivery or distribution of one or more polynucleotides to target cells, tissues and/or organs.
  • lamellar body carriers may also be loaded or otherwise prepared such that they also comprise one or more polynucleotides (e.g., mRNA encoding a functional protein or enzyme).
  • the compositions disclosed herein may comprise synthetically- or naturally-prepared lamellar bodies and lipid nanoparticles.
  • compositions disclosed herein comprise lamellar bodies
  • lamellar bodies may comprise one or more of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), sphingomyelin (SM), cholesterol (CHOL) and dipalmitoylphosphatidylcholine (DPPC).
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • SM sphingomyelin
  • cholesterol CHOL
  • DPPC dipalmitoylphosphatidylcholine
  • compositions and/or carriers disclosed herein may also comprise one or more exosomes.
  • Exosomes are small micro-vesicles that are shed from the surface membranes of most cell types (e.g., mammalian cell types) and that have been implicated as playing a pivotal role in cell-to-cell communications (e.g., as a vehicle for transferring various bioactive molecules).
  • cell types e.g., mammalian cell types
  • cell-to-cell communications e.g., as a vehicle for transferring various bioactive molecules.
  • the liver represents an important peripheral target organ for the compositions of the present invention in part due to its central role in metabolism and production of proteins and accordingly diseases which are caused by defects in liver-specific gene products (e.g., the urea cycle disorders) may benefit from specific targeting of cells (e.g., hepatocytes). Accordingly, in certain embodiments of the present invention, the structural characteristics of the target tissue may be exploited to direct the distribution of the liposomal carrier and its polynucleotide payload to such target tissues.
  • a liposomal carrier may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; accordingly the liposomal carrier can readily penetrate such endothelial fenestrations to reach the target hepatocytes.
  • a liposomal carrier may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues (e.g., peripheral cells and tissues).
  • a liposomal carrier may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the liposomal carrier to hepatocytes.
  • large liposomal carriers will not easily penetrate the endothelial fenestrations, and would instead be cleared by the macrophage Kupffer cells that line the liver sinusoids.
  • the size of the carrier is within the range of about 25 to 250 nm, preferably less than about 250nm, 175nm, 150nm, 125nm, 100nm, 75nm, 50nm, 25nm or 10nm.
  • compositions of the present invention may be prepared to preferentially distribute to other local and/or peripheral target tissues, cells or organs, such as the brain, cerebrospinal fluid, muscle, heart, lungs, kidneys and/or spleen.
  • the carriers of the present invention may be prepared to achieve enhanced delivery to the target cells and tissues.
  • the compositions of the present invention may be enriched with additional cationic, non-cationic and PEG-modified lipids to further target tissues or cells.
  • one or more peripheral target cells and tissues may function as a biological reservoir or depot capable of expressing or otherwise producing and systemically excreting a functional protein or enzyme, as disclosed for example, in International Application No. PCT/US2010/058457 and in United States Provisional Application No. 61/494,881 , the teachings of which are both incorporated herein by reference in their entirety.
  • the liposomal carrier may target cells and/or preferentially distribute to one or more target cells and tissues (e.g., target cells and tissues of the liver) following their delivery to a subject. Following transfection of the target cells (e.g., local endothelial cells of the lung), the nuclease resistant mRNA polynucleotides loaded in the carrier are translated and a functional expression product expressed, excreted and systemically distributed.
  • compositions of the present invention comprise one or more additional molecules (e.g., proteins, peptides, aptamers or oliogonucleotides) which facilitate the transfer of the polynucleotides (e.g., mRNA, miRNA, snRNA and snoRNA) from the carrier into an intracellular compartment of the target cell.
  • the additional molecule facilitates the delivery of the polynucleotides into, for example, the cytosol, the lysosome, the mitochondrion, the nucleus, the nucleolae or the proteasome of a target cell.
  • Such agents may facilitate the transport of the translated protein of interest from the cytoplasm to its normal intercellular location (e.g., in the mitochondrion) to treat deficiencies in that organelle.
  • the agent is selected from the group consisting of a protein, a peptide, an aptamer, and an oligonucleotide.
  • the compositions described herein may comprise one or more excipients that facilitate the distribution of such compositions into the plasma, where such compositions may further distribute to one or more additional target organs, tissues or cells.
  • compositions of the present invention facilitate a subject's endogenous production of one or more functional proteins and/or enzymes.
  • the endogenous production or translation of exogenous nuclease resistant mRNA polynucleotides by a subject to produce one or more expression products may, in certain instances demonstrate less immunogenicity relative to their recombinantly-prepared counterparts that often lack native post-translational modifications (e.g., glycosylation).
  • the endogenously produced or translated proteins and/or enzymes may demonstrate more biological activity relative to their recombinantly-prepared counterparts.
  • the carriers comprise nuclease resistant mRNA polynucleotides which encode a deficient expression product (e.g., a protein or enzyme).
  • a deficient expression product e.g., a protein or enzyme.
  • the administration of an mRNA polynucleotide (e.g., a nuclease resistant mRNA polynucleotide) encoding a deficient protein or enzyme avoids the need to deliver the polynucleotides to specific organelles within a target cell (e.g., mitochondria). Rather, upon transfection of a target cell and delivery of the polynucleotides to the cytoplasm of the target cell, the mRNA polynucleotide contents of a carrier may be translated and a functional protein or enzyme expressed.
  • the present invention also contemplates the discriminatory targeting of target cells and tissues by both passive and active targeting means.
  • the phenomenon of passive targeting exploits the natural distributions patterns of a carrier in vivo without relying upon the use of additional excipients or means to enhance recognition of the carrier by target cells.
  • carriers which are subject to phagocytosis by the cells of the reticulo-endothelial system are likely to accumulate in the liver or spleen, and accordingly may provide means to passively direct the delivery of the compositions to such target cells.
  • the present invention also contemplates active targeting, which involves the use of additional excipients, referred to herein as "targeting ligands" that may be bound (either covalently or non-covalently) to the carrier to encourage localization of such carrier at certain target cells or target tissues.
  • targeting may be mediated by the inclusion of one or more endogenous targeting ligands (e.g., apolipoprotein E) in or on the carrier to encourage distribution to the target cells or tissues.
  • endogenous targeting ligands e.g., apolipoprotein E
  • the composition can comprise a ligand capable of enhancing affinity of the composition to the target cell.
  • Targeting ligands may be linked to the outer bilayer of the lipid particle during formulation or post-formulation.
  • lipid particle formulations may employ fusogenic polymers such as PEAA, hemagluttinin, other lipopeptides (see U.S. Patent Application Ser. Nos. 08/835,281 , and 60/083,294 , which are incorporated herein by reference) and other features useful for in vivo and/or intracellular delivery.
  • fusogenic polymers such as PEAA, hemagluttinin, other lipopeptides (see U.S. Patent Application Ser. Nos. 08/835,281 , and 60/083,294 , which are incorporated herein by reference) and other features useful for in vivo and/or intracellular delivery.
  • the compositions of the present invention demonstrate improved transfection efficacies, and/or demonstrate enhanced selectivity towards target cells or tissues of interest.
  • compositions which comprise one or more ligands (e.g., peptides, aptamers, oligonucleotides, a vitamin or other molecules) that are capable of enhancing the affinity of the compositions and their polynucleotide contents for the target cells or tissues.
  • ligands e.g., peptides, aptamers, oligonucleotides, a vitamin or other molecules
  • Suitable ligands may optionally be bound or linked to the surface of the carrier.
  • the targeting ligand may span the surface of a carrier or be encapsulated within the carrier.
  • Suitable ligands and are selected based upon their physical, chemical or biological properties (e.g., selective affinity and/or recognition of target cell surface markers or features.) Cell-specific target sites and their corresponding targeting ligand can vary widely.
  • compositions of the present invention may bear surface markers (e.g., apolipoprotein-B or apolipoprotein-E) that selectively enhance recognition of, or affinity to hepatocytes (e.g., by receptor-mediated recognition of and binding to such surface markers).
  • surface markers e.g., apolipoprotein-B or apolipoprotein-E
  • the use of galactose as a targeting ligand would be expected to direct the compositions of the present invention to parenchymal hepatocytes, or alternatively the use of mannose containing sugar residues as a targeting ligand would be expected to direct the compositions of the present invention to liver endothelial cells (e.g., mannose containing sugar residues that may bind preferentially to the asialoglycoprotein receptor present in hepatocytes).
  • liver endothelial cells e.g., mannose containing sugar residues that may bind preferentially to the asialoglycoprotein receptor present in hepatocytes.
  • targeting ligands that have been conjugated to moieties present in the carrier (e.g., a lipid nanoparticle) therefore facilitate recognition and uptake of the compositions of the present invention in target cells and tissues.
  • suitable targeting ligands include one or more peptides, proteins, aptamers, vitamins and oligonucleotides.
  • the carriers disclosed herein may also comprise one or more opsonization-inhibiting moieties, which are typically large hydrophilic polymers that are chemically or physically bound to a carrier or vehicle such as a lipid nanoparticle (e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids).
  • opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the pharmaceutical carrier or vehicle (e.g., liposomes) by the macrophage-monocyte system and reticulo-endothelial system, as described for example, in U.S. Pat. No. 4,920,016 , the entire disclosure of which is herein incorporated by reference.
  • Carriers modified with opsonization-inhibition moieties thus remain in the circulation much longer than their unmodified counterparts.
  • the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, to which the compositions and methods of the present invention are administered.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • target cell refers to a cell to which a composition, nuclease resistant polynucleotide and/or stabilizing oligonucleotide of the invention are to be directed or targeted.
  • the target cells are deficient in a protein or enzyme of interest.
  • cells are targeted based on their ability to secrete one or more expression products extracellularly.
  • compositions and methods of the present invention may be prepared to preferentially target a variety of target cells, which include, but are not limited to, pulmonary epithelial cells (e.g., Type I and II pneumocytes), alveolar cells, hepatocytes, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes
  • the target cells comprise Type I pneumocytes, Type II pneumocytes, alveolar cells and combinations thereof.
  • expression of the polypeptide, protein or enzyme encoded by such polynucleotide may be preferably stimulated and the capability of such target cells to express the protein of interest enhanced.
  • transfection of a target cell with a stabilized or duplexed mRNA polynucleotide encoding the OTC enzyme may facilitate the enhanced expression of the corresponding expression product (OTC) following translation of the mRNA polynucleotide.
  • OTC corresponding expression product
  • Such methods generally comprise administering to the subject (e.g., parenterally) a composition comprising a nuclease resistant mRNA polynucleotide and a suitable carrier, wherein the mRNA encodes an enzyme or protein in which the subject is deficient.
  • compositions and methods of the present invention may be suitable for the treatment of diseases or disorders relating to the deficiency of proteins and/or enzymes.
  • the stabilized or nuclease resistant polynucleotides of the present invention encode functional proteins or enzymes that are excreted or secreted by the target cell into the surrounding extracellular fluid (e.g., mRNA encoding hormones and neurotransmitters).
  • the polynucleotides (e.g., mRNA encoding urea cycle metabolic disorders) of the present invention encode functional proteins or enzymes that remain in the cytosol of the target cell.
  • disorders for which the present invention are useful include disorders such as Duchenne muscular dystrophy, blood clotting disorders, such as e.g., hemophelia, SMN1-related spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-related galactosemia; Cystic Fibrosis (CF); SLC3A1-related disorders including cystinuria; COL4A5-related disorders including Alport syndrome; galactocerebrosidase deficiencies; X-linked adrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-related tuberous sclerosis; Sanfilippo B syndrome (MPS IIIB); CTNS-related cystinosis; the FMR1-related disorders which include Fragile X syndrome, Fragile X-Associated Tremor/Ataxia Syndrome and Fragile X Premature Ovarian Failure Syndrome; Pra
  • the polynucleotides, and in particular mRNA, of the present invention may encode functional proteins or enzymes.
  • the compositions of the present invention may include mRNA encoding erythropoietin, ⁇ 1-antitrypsin, carboxypeptidase N, human growth hormone, Factor VII, Factor III, Factor IX, or cystic fibrosis transmembrane conductance regulator (CFTR).
  • CFTR cystic fibrosis transmembrane conductance regulator
  • nuclease resistant polynucleotides disclosed herein may encode full length antibodies or smaller antibodies (e.g., both heavy and light chains) to confer immunity to a subject. While certain embodiments of the present invention relate to methods and compositions useful for conferring immunity to a subject (e.g., via the translation of mRNA polynucleotides encoding functional antibodies), the inventions disclosed herein and contemplated hereby are broadly applicable. In an alternative embodiment the compositions of the present invention encode antibodies that may be used to transiently or chronically affect a functional response in subjects.
  • the nuclease resistant mRNA polynucleotides of the present invention may encode a functional monoclonal or polyclonal antibody, which upon translation (and as applicable, systemic excretion from the target cells) may be useful for targeting and/or inactivating a biological target (e.g., a stimulatory cytokine such as tumor necrosis factor).
  • a biological target e.g., a stimulatory cytokine such as tumor necrosis factor
  • nuclease resistant mRNA polynucleotides of the present invention may encode, for example, functional anti-nephritic factor antibodies useful for the treatment of membranoproliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or alternatively may encode anti-vascular endothelial growth factor (VEGF) antibodies useful for the treatment of VEGF-mediated diseases, such as cancer.
  • VEGF vascular endothelial growth factor
  • compositions of the present invention may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art.
  • the "effective amount" for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts.
  • the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art.
  • a suitable amount and dosing regimen is one that causes at least transient expression of the stable or nuclease resistant polynucleotide in the target cell.
  • Suitable routes of administration of the compositions disclosed herein may include, for example, pulmonary, oral, rectal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • compositions of the present invention are formulated such that they are suitable for extended-release of the stabilized or nuclease resistant polynucleotides contained therein.
  • extended-release compositions may be conveniently administered to a subject at extended dosing intervals.
  • the compositions of the present invention are administered to a subject twice day, daily or every other day.
  • the compositions of the present invention are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, or more preferably every four weeks, once a month, every six weeks, every eight weeks, every other month, every three months, every four months, every six months, every eight months, every nine months or annually.
  • compositions and liposomal carriers which are formulated for depot administration (e.g., intramuscularly, subcutaneously, intravitreally) to either deliver or release a polynucleotides (e.g., mRNA) over extended periods of time.
  • a polynucleotides e.g., mRNA
  • the extended-release means employed are combined with modifications made to the polynucleotide to enhance stability.
  • lyophilized pharmaceutical compositions comprising one or more of the compounds disclosed herein and related methods for the use of such lyophilized compositions as disclosed for example, in United States Provisional Application No. 61/494,882 filed June 8, 2011 , the teachings of which are incorporated herein by reference in their entirety.
  • the lyophilized pharmaceutical compositions according to the invention may be reconstituted prior to their administration to a subject (e.g., reconstituted using purified water or normal saline and inhaled by a subject using a device such as a nebulizer).
  • the lyophilized compositions can be reconstituted in vivo, for example by lyophilizing such composition in an appropriate dosage form (e.g., an intradermal dosage form such as a disk, rod or membrane) and administering such composition such that it is rehydrated over time in vivo by the individual's bodily fluids.
  • an appropriate dosage form e.g., an intradermal dosage form such as a disk, rod or membrane
  • the present example illustrates the ability of stabilizing oligonucleotides of the present invention to enhance the production of protein when co-administered with non-denatured in vitro transcribed mRNA. Without wishing to be bound by any theory, it is contemplated that the stabilizing oligonucleotides modulate the nuclease resistance and increases the translational efficiency of mRNA polynucleotide transcripts.
  • EPO erythropoietin
  • the EPO mRNA transcript was contacted with the stabilizing oligonucleotide at 0.001:1, 0.01:1, 0.1:1, 0.25:1, 1:1, 10:1 and 100:1 parts stabilizing oligonucleotide to mRNA polynucleotide.
  • the resultant stabilized mRNA transcripts (designated “0.001”, “0.01”, “0.1”, “0.25", “1", “10” or “100") or the untreated, non-denatured EPO polynucleotide control transcript (designated “Unhybridized”) were then transiently transfected into 293T cells.
  • the cumulative amounts of EPO protein produced and expressed by the transfected 293T cells were then measured at 6, 24 and 72 hour intervals.
  • the stabilized EPO mRNA transcript prepared using 100:1 parts stabilizing oligonucleotide to mRNA designated "100"
  • the cumulative amount of EPO protein produced and secreted by the 293T cells that were transfected with the stabilized mRNA transcripts exceeded the cumulative amount of EPO protein produced by the cells transfected with the Unhybridized mRNA transcript.
  • the stabilized EPO mRNA transcripts designated 0.001, 0.01, 0.1, 0.25, 1 and 10 each resulted in the production of more EPO protein relative to the Unhybridized EPO control and, in certain instances exceeded the amount of EPO protein produced by the control by over 160% at the 6 hour time point.
  • the present example further illustrates the ability of the stabilizing oligonucleotides of the present invention to enhance the protein production by first hybridizing to a denatured single-stranded mRNA to form a stabilized mRNA before administering into cells for protein production
  • a 15-mer (2'O-Me-uracil) stabilizing oligonucleotide having a phosphorothioate backbone was prepared and which was designed to be complementary to the poly-A tail of an mRNA polynucleotide encoding human erythropoietin (EPO) protein.
  • EPO erythropoietin
  • the EPO mRNA transcript was first denatured at 65°C for 10 minutes, and then contacted with the stabilizing oligonucleotide at 0.001:1, 0.01:1, 0.1:1, 0.25:1, 1:1, 10:1 and 100:1 parts stabilizing oligonucleotide to mRNA polynucleotide.
  • the resultant stabilized mRNA transcripts (designated “0.001”, “0.01”, “0.1”, “0.25", “1", “10” or “100") or the untreated, denatured EPO polynucleotide control transcript (designated “Unhybridized”) were then transiently transfected into 293T cells.
  • the cumulative amounts of EPO protein produced and expressed by the transfected 293T cells were then measured at 6, 24 and 72 hour intervals.
  • the percentage of the cumulative amount of EPO protein produced and secreted by the 293T cells transfected with the stabilized mRNA polynucleotide consistently exceeded the percentage of the cumulative amount of EPO protein produced and secreted by the Unhybridized mRNA polynucleotide at each time point evaluated.
  • the stabilized EPO transcript designated 0.01 demonstrated an approximately 700% increase in the cumulative amount of EPO protein produced relative to the Unhybridized control transcript at the 6 hour time point and in excess of 1,000% at both the 24 hour and 72 hour time points.
  • Each of the stabilized mRNA transcripts evaluated were characterized by an increase in the cumulative amount of EPO protein produced relative to the Unhybridized control.
  • a 30-mer (2'O-Me-uracil) stabilizing oligonucleotide having a phosphorothioate backbone was prepared and which was designed to be complementary to the poly-A tail of an mRNA polynucleotide encoding human erythropoietin (EPO) protein.
  • EPO erythropoietin
  • a non-denatured EPO mRNA transcript was contacted with the stabilizing oligonucleotide at 0.001:1, 0.01:1, 0.1:1, 0.25:1, 0.5:1, 1:1 and 2:1 parts stabilizing oligonucleotide to mRNA polynucleotide.
  • the resultant stabilized mRNA transcripts (designated “0.001”, “0.01”, “0.1”, “0.25", “0.5”, “1” or “2”) or the untreated, non-denatured EPO polynucleotide control transcript (designated “Unhybridized”) were then transiently transfected into 293T cells.
  • the cumulative amounts of EPO protein produced and expressed by the transfected 293T cells were then measured at 24, 48, 72 and 96 hour intervals.
  • those stabilized mRNA polynucleotides prepared using 0.1:1 and 0.5:1 parts stabilizing oligonucleotide to mRNA polynucleotide (designated "0.1” and "0.5"), cumulatively produced and secreted more EPO protein relative to the Unhybridized control polynucleotide.
  • an approximately 10% reduction of the cumulative amount of EPO protein produced relative to the Unhybridized control polynucleotide was observed with several of the stabilized mRNA transcripts evaluated (e.g., the stabilized mRNA transcript designated "0.25").
  • the cumulative amount of EPO protein produced using the 30-mer stabilizing oligonucleotide appeared to be less than that observed using shorter stabilizing oligonucleotides (e.g., a 15-mer stabilizing oligonucleotide).
  • shorter stabilizing oligonucleotides e.g., a 15-mer stabilizing oligonucleotide.
  • such reduction may be due in part to the greater degree of hybridization observed with longer stabilizing oligonucleotides, or the interference with the ability of the mRNA transcript to form stable secondary structures.
  • a greater degree of hybridization of the stabilizing oligonucleotide to the mRNA transcript may interfere (e.g., sterically interfere) with the ability of the mRNA transcript to form secondary structures (e.g., hairpin loops) that may serve to further protect and stabilize the mRNA transcript from nuclease degradation.
  • a greater degree of hybridization of the mRNA transcript may negatively impacting endogenous cellular function, for example, by interfering with the ability of cells or of organelles within such cells to translate the mRNA polynucleotide transcript.
  • the present inventors have also observed that hybridization of the stabilizing oligonucleotides to the mRNA polynucleotide transcript at lower concentrations (in particular at 0.01:1, 0.1: 1, 0.25:1, 0.5:1, 1:1,2:1, 10:1 parts stabilizing oligonucleotide to mRNA polynucleotide) appear to have stabilized the mRNA polynucleotide from nuclease degradation, while not materially impacting or negatively interfering with the ability of such stabilized mRNA transcript to form secondary structures.
  • an mRNA transcript to lower concentrations or ratios of the stabilizing oligonucleotide (e.g., about 0.01:1, 0.1: 1, 0.25:1, 0.5:1, 1:1, 2:1, 10:1 parts stabilizing oligonucleotide to mRNA polynucleotide) therefore appears to provide optimum stabilization of mRNA polynucleotide transcript.
  • the stabilizing oligonucleotides of shorter lengths e.g., about 15-mer

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